Method for identifying compounds or lead structures against rna target motifs and rna/protein interactions

Abstract

Disclosed is a method for identifying compounds (lead structures) which specifically bind to (a) desired RNA target motif and can inhibit or eliminate the function thereof or (b) suppress a compound associated with a desired RNA target motif and can thereby inhibit or eliminate the function thereof. The inventive method is based on the attachment of a ligand (=a compound to be identified) to a RNA target motif which is coupled to a modified ribozyme so that the ribozyme is transformed into an active or inactive conformation resulting in the cleaving of a signal-giving ribozyme substrate. The identified compounds enabling modification of the cellular function of the RNA target motifs enable specific medicaments to be produced. The invention also relates to a polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule. The base pairing model of the polynucleotide, when the target molecule binds to the aptamer, is different from the base pairing model of the polynucleotide when the target molecule does not bind to the aptamer.

1 . A method for identifying compounds which bind specifically to a desired RNA-target motif and, as a result, can inhibit or eliminate the function of this and which has the following characteristics: 
 (a) Generation of a construct (target reporter construct; TRK) from a reporter ribozyme domain (I) and the RNA target motif (II), where (I) and (II) are linked to each other by an RNA linker and where the reporter ribozyme domain (I) changes its catalytic activity after specific binding of a compound to the RNA target motif (II);    (b) Generation of a signaling ribozyme substrate which can specifically bind to the reporter ribozyme domain (I);    (c) Bringing into contact of the TRK from step (a) and the ribozyme substrate from step (b) with the compound to be identified or a mixture which contains this compound; and    (d) Determination of the binding of the compound to the RNA target motif.    
     
     
         2 . A method for identifying compounds which can displace a compound that is associated with a desired RNA target motif and which, as a result, can inhibit or eliminate the function of this, being characterized by the following steps: 
 (a) Production of a construct (target reporter construct; TRK) from a reporter ribozyme domain (I) and the RNA target motif (II), where (I) and (II) are linked to each other by an RNA linker and where the reporter ribozyme domain (I) alters its catalytic activity after displacement of the compound associated with the desired RNA target motif from the RNA target motif (II);    (b) Production of a signaling ribozyme substrate which specifically binds to the reporter ribozyme domain (I);    (c) Bringing into contact of the TRK from step (a) with the compound which is naturally associated with the RNA target motif;    (d) Bringing into contact of the complex from step (c) and the ribozyme substrate from step (b) with the compound which is to be identified or a mixture which contains this compound; and    (e) Determination of the displacement of the compound associated with the RNA target motif.    
     
     
         3 . The method according to claims  1  or  2 , in which the ribozyme substrate can be cleaved by the reporter ribozyme domain (I) in step (b) and in which the binding is determined in the last step by the cleavage of the ribozyme substrate.  
     
     
         4 . The method according to any of  claims 1  to  3 , in which the reporter ribozyme domain originates from a hammerhead ribozyme.  
     
     
         5 . The method according to any of  claims 1  to  4  in which the ribozyme substrate is a doubly labeled ribozyme substrate.  
     
     
         6 . The method according to any of  claims 1  to  5 , in which the doubly labeled ribozyme substrate contains a fluorophore group and a fluorescence quenching group and in which the quenching of the fluorescence of the fluorophore by the fluorescence quenching group is prevented after cleavage of the reporter ribozyme domain.  
     
     
         7 . The method according to  claim 6 , in which the fluorophore group is 6-carboxyfluorescein (FAM) and the fluorescence quenching group is 6-carboxytetramethylrhodamine (TAMRA).  
     
     
         8 . A drug containing a compound which has been identified by a method according to any of  claims 1  to  6 .  
     
     
         9 . A kit for the performance of a method according to any of  claims 1  to  9 , where the kit comprises the following compounds: 
 (a) A target reporter construct; and    (b) A signaling ribozyme substrate.    
     
     
         10 . A polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, where the binding site of the catalytic domain of the ribozyme for a ribozyme substrate is blocked to the binding of the substrate of the ribozyme in the absence of the target molecule of the aptamer.  
     
     
         11 . The polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular according to  claim 10 , which also comprises the target molecule bound to the aptamer, where the binding site of the catalytic domain of the ribozyme for a ribozyme substrate is accessible for the binding of a substrate for the ribozyme in the presence of the target molecule of the aptamer.  
     
     
         12 . The polynucleotide comprising a hammerhead ribozyme and an aptamer for a target molecule, in particular a polynucleotide according to claims  10  or  11 , in which the pattern of base pairing differs, depending on whether the target molecule of the aptamer is bound or absent.  
     
     
         13 . The polynucleotide according to any of  claims 10  to  12  which also comprises a substrate for the catalytic activity of the ribozyme.  
     
     
         14 . The polynucleotide according to  claim 13 , characterized by the substrate being an FRET substrate.  
     
     
         15 . The polynucleotide according to any of  claims 10  to  12 , comprising a nucleic acid sequence which is selected from the group comprising SEQ. ID. No. 51 and SEQ. ID. No 52.  
     
     
         16 . The polynucleotide according to any of  claims 10  to  15 , characterized by the polynucleotide being RNA, DNA or mixtures of these.  
     
     
         17 . A biosensor, comprising a polynucleotide according to any of  claims 10  to  16 .  
     
     
         18 . The biosensor according to  claim 17 , characterized by the polynucleotide being bound to a solid carrier.  
     
     
         19 . A method for identifying a compound which binds to a target molecule, including the following steps: 
 a) Providing a polynucleotide according to any of  claims 10  to  16 ,    b) Optionally determining the catalytic activity of the ribozyme,    c) Adding the target molecule, where the target molecule interacts with the nucleic acid which binds the target molecule,    d) Optionally determining the catalytic activity of the ribozyme,    e) Adding a candidate compound,    f) Optionally determining the catalytic activity of the ribozyme,    g) Providing a substrate for the catalytic activity of the polynucleotide and adding of the substrate to the reaction mixture,    h) Determining the binding of the candidate compound to the target molecule.    
     
     
         20 . The method according to  claim 19 , being characterized by that either before step f) or after step g), the candidate compound is submitted to a procedure, comprising the steps: 
 a) Providing a polynucleotide, whereby the polynucleotide is an allosteric ribozyme and comprises a hammerhead ribozyme and an aptamer which is specific for the target molecule, preferably the aptamer of the polynucleotide according to  claim 19 ,    b) Optionally determining the catalytic activity of the ribozyme,    c) Adding the target molecule, whereby the target molecule interacts with the nucleic acid which binds the target molecule,    d) Optionally determining the catalytic activity of the ribozyme,    e) Addition of the candidate compound,    f) Optionally determining the catalytic activity of the ribozyme,    g) Providing a substrate for the catalytic activity of the polynucleotide and adding the substrate to the polynucleotide, and    h) Determining the binding of the candidate compound to the target molecule.    
     
     
         21 . The method according to  claim 19  or  20 , characterized by the determination of the binding of the candidate molecule to the target molecule being performed by determining the enzymatic activity of the ribozyme.  
     
     
         22 . The method according to  claim 21 , characterized by the substrate containing a fluorophore group and a fluorescence quenching group and the cleavage of the substrate by the catalytic activity of the ribozyme or the catalytic domain preventing or at least reducing the quenching of the fluorescence.  
     
     
         23 . The method according to  claim 22 , characterized by the fluorophore group being 6-carboxyfluorescein and the fluorescence quenching group being 6-carboxytetramethyl rhodamine or Cy 3.  
     
     
         24 . A drug containing a compound which was identified according to any of  claims 19  to  23 .  
     
     
         25 . A kit for the performance of a procedure according to any of  claims 19  to  23 , comprising 
 a) A polynucleotide in accordance with one of  claims 10  to  16 ; and    b) A signaling ribozyme substrate.    
     
     
         26 . Use of coumermycin for the production of a drug for the treatment of HIV and/or FIV.  
     
     
         27 . Use of nosiheptide for the production of a medicine for the treatment of HIV and/or FIV.  
     
     
         28 . Use of patulin for the production of a medicine for the treatment of HIV and/or FIV.